In Vivo Characterization and Application of the PHA Synthase from Azotobacter vinelandii for the Biosynthesis of Polyhydroxyalkanoate Containing 4-Hydroxybutyrate

Abstract

Polyhydroxyalkanoate (PHA) is a biodegradable thermoplastic naturally synthesized by many microorganisms, and the PHA synthase (PhaC) is known to be the key enzyme involved in determining the material properties and monomer composition of the produced PHA. The ability to exploit widely distributed, commonly found soil microorganisms such as Azotobacter vinelandii to synthesize PHA containing the lipase-degradable 4-hydroxybutyrate (4HB) monomer will allow for convenient production of biocompatible and flexible PHA. Comparisons between the A. vinelandii wild type and mutant strains, with and without a surface layer (S-layer), respectively, in terms of gene or amino acid sequences, synthase activity, granule morphology, and PHA productivity, revealed that the S-layer is the sole factor affecting PHA biosynthesis by A. vinelandii. Based on PHA biosynthesis using different carbon sources, the PhaC of A. vinelandii showed specificity for short-chain-length PHA monomers, making it a member of the Class I PHA synthases. In addition, it was proven that the PhaC of A. vinelandii has the inherent ability to polymerize 4-hydroxybutyrate (4HB) and the mediated accumulation of PHA with 4HB fractions ranging from 10 mol% to as high as 22 mol%. The synthesis of biocompatible PHA containing tailorable amounts of 4HB with an expanded range of elasticity and lipase-degradability will enable a wider range of applications in the biomedical field.

Keywords: Azotobacter vinelandii; P(3HB-co-4HB); PHA synthase; S-layer; polyhydroxyalkanoate (PHA); substrate specificity.